Requirements for obtaining unbiased estimates of neuronal numbers in frozen sections

The use of frozen sectioning is a convenient and rapid means of observing t he results obtained using fluorescent retrograde tracers. Quantitation of t hese results using the biased stereological methods currently available can be associated with large errors. A recently developed steroological tool, the optical dissector, provides unbiased and efficient results, however, th e requirements for its use in frozen sections has not previously been estab lished. In this study, a comparison was made of neuron numbers, estimated u sing the optical dissector method, in the motoneuron pool retrogradely labe lled from the rat sciatic nerve with either Fast Blue or Tetramethylrhodami ne dextran (fluoro-ruby) in methacrylate embedded and frozen spinal cord sp ecimens. Despite over 50% shrinkage in the frozen sections, compared with v irtually no shrinkage in the methacrylate sections, no significant differen ce in labelled motoneuron numbers was observed, provided this shrinkage was taken into account. Correction for section shrinkage is therefore essentia l in order to use the optical dissector with confidence to count fluorescen t labelled neurons in frozen tissue. (C) 2000 Elsevier Science B.V. All rig hts reserved.